Role of matrix metalloproteinases in non‐healing venous ulcers

Chronic venous ulceration (CVU) of the lower limbs is a common condition affecting 1% of the adult population in Western countries, which is burdened with a high complication rate and a marked reduction in the quality of life often due to prolonged healing time. Several metalloproteinases (MMPs) such as MMP‐9 together with neutrophil gelatinase‐associated lipocalin (NGAL) appear to be involved in the onset and healing phases of venous ulcer, but it is still unclear how many biochemical components are responsible for prolonged healing time in those ulcers. In this study, we evaluate the role of MMP‐1 and MMP‐8 in long lasting and refractory venous ulcers. In a 2‐year period we enroled 45 patients (28 female and 17 male, median age 65) with CVU. The enroled population was divided into two groups: group I were patients with non‐healing ulcers (ulcers that had failed to heal for more than 2 months despite appropriate treatments) and group II were patients with healing ulcers (ulcers in healing phases). MMP‐1 and MMP‐8 were measured in fluids and tissues of healing and non‐healing ulcers by means of enzyme‐linked immunosorbent assay (ELISA) and Western blot analysis, respectively. In particular the patterns of the collagenases MMP‐1 and MMP‐8 in healing wounds were distinct, with MMP‐8 appearing in significantly greater amounts especially in the non‐healing group. Our findings suggest that MMP‐1, and MMP‐8 are overexpressed in long lasting CVU. Therefore, this dysregulation may represent the main cause of the pathogenesis of non‐healing CVU.     

Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines

Chronic venous ulcer (CVU) represents a dreaded complication of chronic venous disease (CVD). The onset of infection may further delay the already precarious healing process in such lesions. Some evidences have shown that matrix metalloproteinases (MMPs) are involved and play a central role in both CVUs and infectious diseases. Two groups of patients were enrolled to evaluate the expression of MMPs in infected ulcers and the levels of inflammatory cytokines as well as their prevalence. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non‐infected venous ulcers. MMP evaluation and dosage of inflammatory cytokines in plasma and wound fluid was performed by means of enzyme‐linked immunosorbent assay test; protein extraction and immunoblot analysis were performed on biopsied wounds. The first three most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%). In this study, we documented overall higher levels of MMP‐1 and MMP‐8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP‐2 and MMP‐9. We also documented higher levels of interleukin (IL)‐1, IL‐6, IL‐8, vascular endothelial growth factor and tumour necrosis factor‐alpha in patients with infected ulcers with respect to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. The present results could represent the basis for further studies on drug use that mimic the action of tissue inhibitors of metalloproteinases in order to make infected CVU more manageable.     

Evaluation of wound fluid biomarkers to determine healing in adults with venous leg ulcers: A prospective study

Clinical practice guidelines recommend using repeated wound surface area measurements to determine if a chronic ulcer is healing. This results in delays in determining the healing status. This study aimed to evaluate whether any of a panel of biomarkers can determine the healing status of chronic venous leg ulcers. Forty‐two patients with chronic venous leg ulcers had their wound measured and wound fluid collected at weekly time points for 13 weeks. Wound fluid was analyzed using multiplex enzyme‐linked immunosorbent assay to determine the concentration of biomarkers in the wound fluid at each weekly time point. Healing status was determined by examining the change in wound size at the previous and subsequent weeks. Predictive accuracy with 95% confidence intervals (CI) is reported. Of 42 patients, 105 evaluable weekly time points were obtained, with 32 classified as healing, 27 as nonhealing, and 46 as indeterminate. Thirteen biomarkers significantly differed between healing and nonhealing wounds (p < 0.1) and were included in a multivariate logistic regression model. Granulocyte macrophage‐colony stimulating factor (p < 0.001) and matrix metalloprotease‐13 (p = 0.004) were the best predictors of wound healing. Receiver operating characteristic curves indicated 92% accuracy (95% CI: 85%,100%) for granulocyte macrophage‐colony stimulating factor, and 78% accuracy (95% CI: 65%,90%) for matrix metalloprotease‐13 in discriminating between healing and nonhealing wounds. This study found that two biomarkers from wound fluid can predict healing status in chronic venous leg ulcers. These findings may lead to the ability to determine the future trajectory of a wound and the ability to modify treatment accordingly.     

A pilot study evaluating non‐contact low‐frequency ultrasound and underlying molecular mechanism on diabetic foot ulcers

Non‐contact low‐frequency ultrasound (NCLF‐US) devices have been increasingly used for the treatment of chronic non‐healing wounds. The appropriate dose for NCLF‐US is still in debate. The aims of this pilot study were to evaluate the relationship between dose and duration of treatment for subjects with non‐healing diabetic foot ulcers (DFUs) and to explore the correlation between wound healing and change of cytokine/proteinase/growth factor profile. This was a prospective randomised clinical study designed to evaluate subjects with non‐healing DFUs for 5 weeks receiving standard of care and/or NCLF‐US treatment. Subjects were randomly assigned to one of the three groups: application of NCLF‐US thrice per week (Group 1), NCLF‐US once per week (Group 2) and the control (Group 3) that received no NCLF‐US. All subjects received standard wound care plus offloading for a total of 4 weeks. Percent area reduction (PAR) of each wound compared with baseline was evaluated weekly. Profiles of cytokines/proteinase/growth factors in wound fluid and biopsied tissue were quantified to explore the correlation between wound healing and cytokines/growth factor expression. Twelve DFU patients, 2 (16·7%) type 1 and 10 (83·3%) type 2 diabetics, with an average age of 58 ± 10 years and a total of 12 foot ulcers were enrolled. Average ulcer duration was 36·44 ± 24·78 weeks and the average ABI was 0·91 ± 0·06. Group 1 showed significant wound area reduction at weeks 3, 4 and 5 compared with baseline, with the greatest PAR, 86% (P < 0·05); Groups 2 and 3 showed 25% PAR and 39% PAR, respectively, but there were no statistically significant differences between Groups 2 and 3 over time. Biochemical and histological analyses indicated a trend towards reduction of pro‐inflammatory cytokines (IL‐6, IL‐8, IL‐1β, TNF‐α and GM‐CSF), matrix metalloproteinase‐9 (MMP‐9), vascular endothelial growth factor (VEGF) and macrophages in response to NCLF‐US consistent with wound reduction, when compared with control group subjects. This proof‐of‐concept pilot study demonstrates that NCLF‐US is effective in treating neuropathic diabetic foot ulcers through, at least in part, inhibiting pro‐inflammatory cytokines in chronic wound and improving tissue regeneration. Therapeutic application of NFLU, thrice (3) per week, renders the best wound area reduction.     

Doxycycline speeds up healing of chronic venous ulcers

Venous ulcers are common, with an overall prevalence of up to 2% in the general population of western countries, and have significant socioeconomic impact. Matrix metalloproteinases (MMPs) are involved in the alteration of extracellular matrix that could lead to venous ulceration. Sixty‐four patients with venous ulcers were recruited in a 22‐month period. All patients were subjected to the most appropriate treatment considering also the patient's wishes (compression therapy followed or not by vein surgery). Patients were randomised into two groups of 32 persons in each (groups A and B). Patients of group A in addition to the basic treatment, described above, received the administration of oral low doses of doxycycline 20 mg b.i.d. for 3 months, whereas patients of group B received basic treatment only. Healing was assessed by means of direct ulcer tracing with computerised planimetry. Group A showed a higher healing rate compared with group B. In group B, the lower healing rate was related to higher levels of MMP‐9; neutrophil gelatinase‐associated lipocalin and vascular endothelial growth factor, documented in plasma; wound fluid and biopsies executed and compared between both groups. Pharmacological treatments, as doxycycline administration, which by means of its immunomodulatory and anti‐inflammatory actions, through the inhibition of MMP, could improve extracellular matrix functioning and represent a possible solution to support wound healing.     

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.     

Iliac vein recanalisation and stenting accelerate healing of venous leg ulcers associated with severe venous outflow obstruction

Obstruction involving the iliac veins and/or inferior vena cava is highly comorbid in patients with chronic venous leg ulcers and is a barrier to healing. Intervention with venous stenting is recommended to promote wound healing; however, there is limited data to quantify the effects of venous outflow restoration on wound healing. We retrospectively identified patients with venous ulcers and comorbid venous outflow obstruction. Data regarding demographics, wound size, degree of obstruction, interventions, wound healing and recurrence were collected. Intervention was performed when possible and patients were grouped based on whether or not the venous outflow was reopened successfully and maintained for at least 1 year. Outcomes, including time to wound healing, wound recurrence, stent patency and ulcer-free time, were measured. Patients who maintained a patent venous outflow tract experienced higher rates of wound healing (79.3%) compared to those with persistent outflow obstruction (22.6%) at 12 months (p < 0.001). Ulcer-free time for the first year was also greater with patent venous outflow (7.6 ± 4.4 months versus 1.8 ± 3.0 months, p < 0.0025). Patients with severe obstruction of the venous outflow tract experience poor healing of VLUs despite appropriate wound care. Healing time is improved and ulcer-free time increased after venous intervention with stenting to eliminate obstruction.     

Analysis of pressure ulcer wound fluid using two‐dimensional electrophoresis

The incidence rate of pressure ulcers in the USA ranges from 0·4% to 38% in acute care settings and from 2·2% to 23·9% in long‐term care settings, and their treatment costs are in the billions of dollars yearly. The proteome of wound fluid may contain early indicators or biomarkers associated with healing in pressure ulcers that would enable treatment regimes to be optimised for each individual. Wound fluid was collected from the interior and periphery of 19 chronic pressure ulcers at 15 time points during 42 days for an analysis of protein expression. Proteins were fractionated using two‐dimensional polyacrylamide gel electrophoresis. A comparison of the spot distributions indicates a biochemical difference between the interior and the periphery of wounds. Pressure ulcers that healed show a greater number of spots for interior and peripheral locations combined over time when compared with wounds that did not heal. Using this technique, protein S100A9 was identified as a potential biomarker of wound healing. The identification of differences within the proteome of healing versus non healing pressure ulcers could have great significance in the use of current treatments, as well as the development of new therapeutic interventions.     

Safety and performance evaluation of a next‐generation antimicrobial dressing in patients with chronic venous leg ulcers

The objective of this study was to investigate the safety and performance of AQUACEL? Ag+ dressing, a wound dressing containing a combination of anti‐biofilm and antimicrobial agents, in the management of chronic wounds. Patients (n = 42) with venous leg ulcers exhibiting signs of clinical infection were treated for 4 weeks with AQUACEL? Ag+ dressing, followed by management with AQUACEL? wound dressings for 4 weeks. Wound progression, wound size, ulcer pain and clinical evolution of the wound were assessed for up to 8 weeks. Adverse events were recorded throughout the study. AQUACEL? Ag+ dressing had an acceptable safety profile, with only one patient discontinuing from the study, because of a non‐treatment‐related adverse event. After 8 weeks, substantial wound improvements were observed: 5 patients (11·9%) had healed ulcers and 32 patients (76·2%) showed improvement in ulcer condition. The mean ulcer size had reduced by 54·5%. Patients reported less pain as the study progressed. Notable improvements were observed in patients with ulcers that were considered to require treatment with systemic antibiotics or topical antimicrobials at baseline (n = 10), with a mean 70·2% reduction in wound area. These data indicate that AQUACEL? Ag+ dressing has an acceptable safety profile in the management of venous leg ulcers that may be impeded by biofilm.     

EPA + DHA supplementation reduces PMN activation in microenvironment of chronic venous leg ulcers: A randomized,double‐blind,controlled study

Sustained high levels of activated polymorphonuclear leukocytes (PMNs) and PMN‐derived proteases in the microenvironment of chronic venous leg ulcers (CVLUs) are linked to chronic inflammation and delayed healing. Uncontrolled PMN activity eventually destroys newly developed tissue and degrades critical growth factors. The bioactive components of fish oil (n‐3 eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) have strong inflammation‐resolving actions and have been shown to assuage PMN activity, but have not been tested in CVLU patients. This randomized controlled study compared the effectiveness of oral EPA + DHA therapy to a placebo for reducing PMN activation in CVLU microenvironments. At Days 0, 28, and 56, markers of PMNs (CD15) and activated PMNs (CD66b), and levels of PMN‐derived proteases human neutrophil elastase and matrix metalloproteinase‐8 were measured in CVLU fluid from patients receiving standard compression therapy and (1) EPA + DHA therapy (n = 16) or (2) placebo (n = 19). By Day 56, the EPA + DHA Group had a significantly lower percentage of CD66b+ cells in CVLU fluid compared to Day 0 (p = 0.02) and to Day 28 (p = 0.05). Importantly, there were downward trends in levels of both matrix metalloproteinase‐8 and human neutrophil elastase over time in the EPA + DHA Group, which also demonstrated greater reductions in wound area by Day 28 (57% reduction) and Day 56 (76% reduction) than the Control Group (35% and 59%, respectively). Moreover, reductions in wound area had significant negative relationships with CD15+ cells in wound fluid at Days 28 (p = 0.008) and 56 (p < 0.001), and CD66b+ cells at Days 28 (p = 0.04) and 56 (p = 0.009). The collective findings provide supplemental evidence that high levels of activated PMNs in CVLU microenvironments inhibit healing, and suggest that EPA + DHA oral therapy may modulate PMN activity and facilitate healing of CVLUs when added to standard care regimens.     

A single‐arm trial indirect comparison investigation: a proof‐of‐concept method to predict venous leg ulcer healing time for a new acellular synthetic matrix matched to standard care control

To compare data on time to healing from two separate cohorts: one treated with a new acellular synthetic matrix plus standard care (SC) and one matched from four large UK pragmatic, randomised controlled trials [venous leg ulcer (VLU) evidence network]. We introduce a new proof‐of‐concept strategy to a VLU clinical evidence network, propensity score matching and sensitivity analysis to predict the feasibility of the new acellular synthetic matrix plus SC for success in future randomised, controlled clinical trials. Prospective data on chronic VLUs from a safety and effectiveness study on an acellular synthetic matrix conducted in one wound centre in the UK (17 patients) and three wound centres in Australia (36 patients) were compared retrospectively to propensity score‐matched data from patients with comparable leg ulcer disease aetiology, age, baseline ulcer area, ulcer duration, multi‐layer compression bandaging and majority of care completed in specialist wound centres (average of 1 visit per week), with the outcome measures at comparable follow‐up periods from patients enrolled in four prospective, multicentre, pragmatic, randomised studies of venous ulcers in the UK (the comparison group; VLU evidence network). Analysis using Kaplan–Meier survival curves showed a mean healing time of 73·1 days for ASM plus SC (ASM) treated ulcers in comparison with 83·5 days for comparison group ulcers treated with SC alone (Log rank test, χ² 5·779, P = 0·016) within 12 weeks. Sensitivity analysis indicates that an unobserved covariate would have to change the odds of healing for SC by a factor of 1·1 to impact the baseline results. Results from this study predict a significant effect on healing time when using a new ASM as an adjunct to SC in the treatment of non‐healing venous ulcers in the UK, but results are sensitive to unobserved covariates that may be important in healing time comparison.     

Diabetic wound healing in a MMP9‐/‐ mouse model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.     

From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases

Chronic venous disease (CVD) and its most frightening complication, chronic venous ulceration (CVU), represent an important socioeconomic burden in the western world. Metalloproteinases have been identified in the pathogenesis of several vascular diseases such as venous problems. The aim of this study was to evaluate a broad range of metalloproteinases, such as matrix metalloproteinases (MMPs), ADAMs (a disintegrin and metalloproteinases) and ADAMTSs (a disintegrin and metalloproteinases with thrombospondin motifs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) and a related protein, neutrophil gelatinase‐associated lipocalin (NGAL), in patients with CVD in order to correlate their serum levels with each stage of the disease. We performed a multicenter open‐label study that comprised the enrolment of 541 patients with CVD of clinical stages C1–C6, (178 males, 363 females; mean age 57·29, median age 53·72, age range 29–81); 29 subjects without CVD were included in this study (9 males and 20 females; mean age 54·44, median age 50, age range 28–84) as the control group. Enzyme‐linked immunosorbent assay (ELISA) was performed for measuring serum levels of proteases and related proteins. The study found that the serum elevation of MMP‐2, ADAMTS‐1 and ADAMTS‐7 appeared to be correlated with the initial stages of CVD, whereas the serum elevation of MMP‐1, MMP‐8, MMP‐9, NGAL, ADAM‐10, ADAM‐17 and ADAMTS‐4 was particularly involved in skin change complications. This study showed that each stage of CVD may be described by particular patterns of metalloproteinases, and this may have therapeutic implications in discovering new targets and new drugs for the treatment of CVD.     

Autologous platelet‐rich fibrin matrix as cell therapy in the healing of chronic lower‐extremity ulcers

A novel autologous platelet‐rich fibrin matrix membrane (PRFM) was assessed for the ability to facilitate healing in patients with chronic lower‐extremity ulcers. Preliminary data are presented from a prospective trial (n=21). Twelve patients were identified with 17 venous leg ulcers (VLU) and nine bearing 13 nonvenous lower‐extremity ulcers. Before enrollment, the patients were evaluated for vascular status and received appropriate surgical intervention to optimize arterial and venous circulatory status. None of the ulcers had responded to a variety of standard treatments from 4 months to 53 years. Initial ulcer size ranged from 0.7 to 65 cm² (mean, 11.2 cm²). Each PRFM‐treated patient received up to three applications of either a 35 or 50 mm fenestrated membrane, depending on initial ulcer size. The primary endpoints were percent and rate of complete closure as measured by digital photography, computerized planimetery, and clinical examination. Patients were followed weekly for 12 weeks with a follow‐up visit at 16 weeks. At each 4‐week interval, the extent of healing was assessed, and those patients with >50% reduction in wound area were allowed to continue to complete closure. Patients with <50% closure received repeated applications. Complete closure was achieved in 66.7% of the VLU patients (64.7% of treated ulcers) in 7.1 weeks (median, 6 weeks) with an average of two applications per patient. Forty‐four percent complete closure was seen with non‐VLU patients (31% of treated ulcers). From the results of this small‐scale pilot study, PRFM shows significant potential for closing of chronic leg ulcers.     

Efficacy and cost‐effectiveness of octenidine wound gel in the treatment of chronic venous leg ulcers in comparison to modern wound dressings

The aim of this study was to determine the efficacy, safety and cost‐effectiveness of an octenidine‐based wound gel in the treatment of chronic venous leg ulcers. For this purpose, 49 wounds were treated with either modern wound‐phase‐adapted dressings alone (treatment arm 1; n = 17), octenidine wound gel plus modern wound‐phase‐adapted dressings (treatment arm 2; n = 17) or octenidine wound gel alone (treatment arm 3; n = 15). During the study period of 42 days with dressing changes every 3–5 days, wound healing characteristics and treatment costs of different dressings were analysed. Wound size reduction was significantly better (P = 0·028) in both octenidine wound gel treatment arms compared to modern dressings alone with total reductions of 14·6%, 64·1% and 96·2% in treatment arms 1–3. Early wound healing was merely observed under octenidine wound gel treatment (n = 9), whereby lowest treatment costs were generated by octenidine wound gel alone (€20·34/dressing change). As a result, the octenidine wound gel is cost‐effective and well suitable for the treatment of chronic venous leg ulcers, considering both safety and promotion of wound healing.     

Does localized iron loss in venous disease lead to systemic iron deficiency? A descriptive pilot study

Haemosiderin deposition in the legs of patients with venous leg ulcers is well established, and several theories suggest this stored iron has a role in disease pathophysiology. In this novel pilot study of patients with chronic venous leg ulcers, we aimed to establish the relationship between wound fluid iron levels, serum iron parameters and healing. Fifteen patients with venous ulcers were included in the study. Blood samples were taken for full blood count and iron studies, while simultaneously wound fluid was obtained from the wound surface using filter paper. Wound areas were measured at initial and 4 week (+/? 2 day) follow‐up visits. We found a positive correlation between wound fluid and serum iron (correlation co‐efficient 0.27) and those with the lowest wound fluid iron level were also anemic. No association was found between initial wound area and wound fluid iron level but the largest wound areas were found in patients with anemia. Only 38% of patients demonstrated a reduction in wound area during the 4 week study, and 80% of those were not anemic or iron deficient. Conversely in those patients whose wounds did not reduce in size 88% were anemic or iron deficient. These findings demonstrate a previously unrecognized phenomenon of systemic iron store depletion secondary to leaching out of the body in wound exudate. In addition, these results suggest a high prevalence of anemia in patients with chronic venous ulcers, though whether this is cause or effect requires further research. Our findings also suggest that patients with venous ulcers have a high prevalence of iron deficiency and anemia, which appears to be often undiagnosed, and that diagnostic criteria for iron deficiency in patients with chronic wounds need to be revised to reflect the effect of chronic inflammation on iron metabolism.     

Chemokine and inflammatory cytokine changes during chronic wound healing.

Wound healing is a complex process resulting from an interplay of processes including coagulation, inflammation, angiogenesis, and epithelialization. The chemokine family has been shown to contain members that are potent regulators of many of these pathways. Because we have previously shown that chemokines "pool" in biologic wound dressings, we studied the levels of CXC and CC chemokines, along with key inflammatory mediators, serially from a group of patients undergoing therapy for chronic venous leg ulcers. After 8 weeks, all patients had marked clinical healing of their ulcers (median 63.3% reduction in size) with two of 10 completely healed. Wound fluids extracted from dressings showed high levels of platelet factor-4 and interferon-gamma-inducible protein, with a trend toward increases in the ratio of the sums of the angiogenic versus angiostatic CXC chemokines (p = 0.082) in the tissues collected from the center of the ulcers during wound closure. Neutrophil-activating peptide-2 and interleukin-8 accounted for the most changes in wound fluid angiogenic chemokines, with significant differences both as compared with baseline levels and with patients' plasma level noted at various time points between weeks 0 and 8. The level of angiostatic chemokines, interferon-y inducible protein 10 and platelet-activating-4, fell most significantly between weeks 0 and 3 as compared with plasma levels. The observed shift toward angiogenic CXC chemokines suggests that effective healing in chronic venous insufficiency ulcers appears to "move" the ulcer bed toward a state more conducive to epithelialization,characteristic of the proliferative phase of wound healing. CC chemokines were also elevated at baseline in the wound fluid samples as compared with the patients' plasma levels. Macrophage inflammatory protein-1 (3 and regulated on activation, normal T expressed and secreted (RANTES) levels decreased with healing, whereas there were significant increases in the tissue levels of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 a over the first 4 weeks of therapy (p< or = 0.05 for both). Coincident with these changes was a steady increase in the ratio of interleukin-1 R/interleukin-1 receptor antagonist protein in the ulcer center tissues, which also correlated with healing (p < 0 .05) as compared with a decreasing ratio at the ulcer edge, and a biphasic response in the wound fluids. These findings suggest that advanced wound care techniques help move the ulcer from a chronic inflammatory state into one more characteristic of the late inflammatory/early proliferative phase of wound healing. Chemokines may play a critical role in the pathogenesis of chronic venous ulcers through their effects on angiogenesis and/or the progression of inflammatory reactions at the site of injury.     

The impact of differential expression of extracellular matrix metalloproteinase inducer, matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-2 and PDGF-AA on the chronicity of venous leg ulcers.

INTRODUCTION: Alteration in the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase-2 (MMP-2), tissue inhibitors of matrix metalloproteinases (TIMP-2) and platelet derived growth factor (PDGF-AA) may contribute to poor healing in venous leg ulcers. AIM: The aim of this study is to determine the expression of EMMPRIN, MMP-2, TIMP-2 and PDGF-AA in the ulcer exudates and perivascular tissue of healing and non-healing chronic venous ulcers. PATIENTS, MATERIALS AND METHODS: Forty patients with chronic venous ulcers were included in this study, with a mean age of 60 years. Eleven patients were males and 29 were females. All patients had normal ankle brachial index and a venous ulcer of at least 8 weeks duration. Immuno-histochemistry using monoclonal antibodies to PDGF-AA, MMP-2, TIMP-2 and EMMPRIN was carried out on paraffin embedded punch biopsy skin specimens from the ulcer edge. Enzyme linked immunosorbent assay for PDGF, MMP-2 and TIMP-2 were carried out on wound fluids collected from patients. The ulcer size and character at the initial assessment and after 8 weeks were assessed to determine the status of ulcer healing. RESULTS: No significant difference was seen in the expression of TIMP-2, MMP-2 and EMMPRIN between the two groups. However, in the non-healing group high levels of MMP-2 and low levels of TIMP-2 in the wound fluid suggest a strong correlation of these two markers in the state of healing. Analysis of wound fluid by ELISA demonstrated high PDGF-AA in the healing group (p = 0.021). Significantly increased levels of PDGF-AA (p<0001) was noted in the perivascular area on immuno-histochemistry of healing ulcers. These data suggest that PDGF-AA plays an important role in healing of venous ulcers. CONCLUSION: Non-healing venous ulcers are associated with greater activity MMP-2 activity. The ratio of MMPs to their inhibitors TIMPs, dictate the rate of healing of the ulcers. PDGF-AA activity is associated with ulcer healing, though the mechanism is unclear. EMMPRIN expression in chronic venous ulcers probably parallels the chronicity of the condition rather than propagate it. However, further studies with larger samples are needed.